Background: The Repeat Expansion Diseases are caused by the intergenerational expansion of a specific tandem repeat. Expansion of a CGG/CCG-repeat in the 5' UTR of the FMR1 gene is associated with 3 different clinical presentations: Individuals with 60-200 repeats, so-called premutation alleles, are at risk for Fragile X-associated tremor-ataxia syndrome (FXTAS). In addition to symptoms resulting from cerebellar degeneration, individuals with FXTAS can have a late-onset dementia, elevated risk of diabetes and hypothyroidism, as well as bowel and urinary incontinence. Female carriers of premutation alleles are also at risk of primary ovarian insufficiency (FXPOI). Since the carrier frequency of these alleles is 1/250 these may be significant public health issues. In addition to these problems, the premutation allele can undergo further expansion on maternal transfer resulting in alleles with >200 repeats. Individuals who inherit these so-called full mutation alleles almost always have Fragile X mental retardation syndrome (FXS), the most common cause of mental retardation and the most common known cause of autism. [unreadable] [unreadable] Progress report: We have generated FXS premutation mice containing 120 CGG/CCG-repeats in the 5 UTR of the endogenous murine Fmr1 gene (Entezam et. al., 2007). Like humans with the same number of repeats, these mice produce elevated levels of Fmr1 mRNA that recent data, from my group (Handa, Saha and Usdin, 2003) and elsewhere, suggests is toxic. These mice also show pathological changes including the accumulation of ubiquitin and lamin A/C positive intranuclear neuronal inclusions in brain. These pathological changes are reminiscent of those seen in human carriers of premutation alleles and suggest that these animals will be suitable for studying the repeat-mediated RNA pathology thought to be responsible for FXTAS. These mice also show evidence of abnormalities in ovarian histopathology that suggest they may also be good models for the ovarian pathology seen in female carriers of premutation alleles. [unreadable] [unreadable] We have identified a number of proteins from mouse brain and ovary whose expression or localization are altered in Fmr1 premutation mice and human premutation carriers. Further characterization of these proteins and their contribution to FXTAS and FXPOI is currently underway. In addition we have shown that the Fmr1 gene itself is abnormally spliced in both human premutation and full mutation carriers and in Fmr1 premutation and KO mice. The splicing changes result in an increase in the production of splice isoforms that have lost exon 14. Since this exon contains the proteins nuclear export signal, this may result in increased expression of FMRP in the nucleus. Since these proteins also have an altered C-terminus, this protein may have properties quite different from that reported for the better studied cytoplasmic isoforms. Work is in progress to understand the mechanism responsible for these splicing changes and the possible consequences this may have for cells in which these isoforms are expressed.[unreadable] [unreadable] Preliminary results from behavioral studies in our premutation mice carried out by one of our collaborators, Dr Carolyn Beebe Smith (NIMH), suggest that these mice have behavioral changes reminiscent of what has been found in mice lacking FMRP. We have previously shown that the premutation mice also have significant regional deficiencies in FMRP in the brain. This is thought to be related to difficulties translating RNAs with large CGG-repeat tracts due to the formation of stable RNA hairpins (Handa, Saha and Usdin, 2003). In particular, the amygdala seems to have relatively low FMRP levels even in young animals. While a inverse relationship between repeat number and FMRP has been seen in human lymphocytes, the etiology of behavioral and cognitive problems in human premutation carriers has been controversial. Our data suggest that repeat-mediated effects on FMRP translation may be responsible for some of the symptoms seen in premutation carriers.